Scientists are developing techniques for analyzing changes in biological and chemical systems, where these changes often relate to the switching between two or more states. For example, Witters et al. in Digital Biology and Chemistry (DOI: 10.1039/C4LC00248B, (Frontier) Lab on a Chip, 2014, 14, pp. 3225-3232) discuss the development of various digital biological and chemical technologies. These digital technologies can work quite well, as digital techniques offer advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. However, digital techniques can be relatively complex, in part due to the technical difficulty in isolating and manipulating single molecules. For example, some techniques use micron-sized magnetic beads to process samples of femtoliter volumes. See Rissin et al., in Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations (DOI:10.1038/nbt.1641, Nature Biotechnology 2010, 28, pp. 595-599). Other techniques use even smaller volumes of attoliters. These tiny volumes can create challenges because the fluid dynamics of small volumes present behaviors, at typical laboratory temperature and pressure that make processing difficult.
For example, most digital detection techniques rely on the micro-compartmentalization of a liquid containing analytes and various detection- and capture-probes. The analytes and detection/capture probes are carried, or exist within, micron-sized droplets, typically of pico-to-attoliter volumes.
Therefore, the way to partition the sample in to smaller volumes is the most important part of a digital detection process. The most readily available device format relies on solid or polymeric substrates forming an array of micro-compartments into which the sample can be transferred. These arrays mainly come in two varieties; (i) the micro-well arrays and (ii) the capillary arrays. In a micro-well array, the compartment is made up by a recess in the substrate, whereas in a capillary array the compartment extends all the way through the substrate, thus forming a through hole. A major challenge inherent in both of these array types is the way that they are loaded with sample and accessory reagents. In the micro-well array, the recess may not readily be filled up with a liquid sample, because air cannot leave the well due to the microscopic dimensions of the well, as an example of this see the research article by Kim et al. entitled “Large-scale femtoliter droplet array for digital counting of single biomolecules” published in Lab on a Chip, (2012) vol. 12, pp. 4986-4991 (DOI: 10.1039/c2Ic40632b). This problem is absent from capillary arrays, because each compartment has two openings, such that if the liquid sample is added from the top opening, then air can escape through the bottom opening. However, when it comes to exchanging the liquid held within the micro-well or capillary compartments with another liquid, an additional issue arises, which is caused by the slow diffusion of molecules. Because both the micro-wells and capillaries are positioned perpendicular to the flow of the liquid phase being added, then good mixing cannot take place, and hence liquid exchange can only take place by molecular diffusion from the bulk liquid into the capillary and vice versa. Consequently, to ensure proper liquid exchange a time-delay (the length of which will depend on the dimensions of the micro-wells/capillaries and the type of molecular species being added) will have to be applied.
To overcome these challenges a third kind of array has been developed, which will be referred to as surface-tension arrays. A surface-tension array is planar and consists of hydrophilic features patterned in or onto a hydrophobic substrate. When a surface-tension array is contacted with an aqueous sample (e.g. by immersion into the aqueous phase and withdrawal of the array) individual droplets may form on the hydrophilic features due to the surface-tension difference between the features and the surrounding substrate. Because the droplets rest on a planar surface, then liquid loading as well as liquid exchange may take place instantaneously (or at least several orders of magnitude faster than for diffusion-limited transport) when a liquid sample is introduced on the array. Unlike the micro-well array, no air can be trapped beneath the liquid and the hydrophilic features and since the array does not rely on depressions/recesses/cavities in the substrate, then liquid mixing between droplets and the bulk liquid is not limited by molecular diffusion. However, all three types of micro-compartmentalization formats (micro-well, capillary and surface tension arrays) are facing the challenge of preserving a large number of liquid micro-droplets for a sufficient long time in order to allow digital counting to be conducted.
At typical ambient temperature and pressure for a laboratory, these microdroplets evaporate within seconds, see for example the research article by Birdi, K. S., Vu, D. T. and Winter, A. entitled “A study of the evaporation rates of small water drops placed on a solid surface” published in The Journal of Physical Chemistry, 1989, vol. 93, pp. 3702-3703 (DOI: 10.1021/j100346a065).
Once evaporated, the ability to process the molecule within the microdroplet is gone, the digital technique cannot be carried out.
Accordingly, it is necessary to prevent rapid evaporation and maintain the microdroplet of a period of time sufficient to measure for the presence of the molecule of interest.
To this end, scientists and engineers have developed certain techniques that seal the compartments that are holding the microdroplets. These seals prevent the microdroplets from contacting the ambient environment and thus prevent evaporation.
There are in general two techniques for sealing a compartment: a physical seal and a chemical seal. The physical seal is used when the compartments are structured as micro-recesses or micro-cavities in a substrate. To physically seal the compartments, an air-tight lid is attached on top of the compartments. In this way, the content of individual compartments cannot evaporate and neighboring compartments cannot exchange their content, which would otherwise lead to cross-contamination. The disadvantage of having a physical seal is that once the compartments have been sealed off, the analysis ends, because the lid cannot be easily removed without disrupting the integrity of the micro-compartments. Furthermore, to apply a physical seal, the compartments have to be structured as micro-wells/-cavities/-recesses, which, due to slow molecular diffusion, results in technical difficulties with exchanging the liquid in the compartments during the initial preparative steps.
One type of chemical seal relies on covering the compartments with an oil (or non-polar liquid) phase. In this way, evaporation of the sample is reduced, because water from the sample only slowly partitions into the oil phase. The advantage of a chemical seal is that it is based on interfacial tension, and hence the compartments do not need to be structured as cavities, but can instead be formed as droplets resting on a surface. This feature enables fast reagent exchange, which is not limited by molecular diffusion, but is instead determined by the flowrate at which the new reagent is introduced. Furthermore, unlike the physical seal, the chemical seal may be removed more easily by aspirating the oil phase from the sample. However, one of the disadvantages of a chemical seal is that analytes or other biomolecules from the sample may partition into the non-polar phase and lead to (i) sample loss and/or (ii) inter-droplet contamination. In particular, biomolecules such as proteins, are prone to be soluble in non-polar liquids, mainly due to the fact that hydrophobic amino acids in the protein may rearrange themselves upon exposure to a hydrophobic interface. This property of molecules to partition from water into a non-polar phase is described by the partition coefficient, i.e. oil-water partition coefficient, water-octanol partition coefficient, etc, e.g. in Lien, E. J. and Ren, S. S. in Chapter 186 in Encyclopedia of Pharmaceutical Technology, Third Edition, 2006, ISBN: 9780849393990. Furthermore, it has been shown that even water—although slowly—partitions into a surrounding oil phase, e.g. see the work of Huebner, A. et al published in Lab on a Chip, 2009, vol. 9, pp. 692-698 (DOI: 10.1039/B813709A). Even further, when a bulk aqueous phase is displaced by a bulk oil phase or vice versa there is a risk of producing emulsion droplets, i.e. micron-sized inclusions of water in oil or vice versa. Emulsion droplets may constitute an experimental nuisance, since they can foul the surfaces and/or deteriorate the flow-performance of the device.
WO2015061362 A1 entitled “Enrichment and detection of nucleic acids with ultrahigh sensitivity” describes how to prepare a non-sealed surface-tension array of liquid droplets exhibiting a fast evaporation rate. WO2013110146 A2 entitled “Patterning device” describes how to prepare a surface tension array of liquid droplets and how to use it for bioassays under a chemical seal. WO02013063230 A1 entitled “Device and method for apportionment and manipulation of sample volumes” describes methods for preparing and using chemically sealed surface-tension arrays for bioassays including digital counting measurements. JP2014021025A entitled “Apparatus and method for forming artificial lipid membrane” describes how to prepare a surface-tension array chemically sealed with a lipid membrane. WO2010039180 A2 entitled “High sensitivity determination of the concentration of analyte molecules or particles in a fluid sample” describes digital counting of analytes by dividing a sample into physically sealed micro-well compartments. WO2010019388 A2 entitled “Method and apparatus for discretization and manipulation of sample volumes” describes micro-well compartments, which may be used to capture and divide a liquid sample by applying a chemical seal comprised by one or more immiscible liquids. WO2012022482 A1 entitled “Microwell arrays for direct quantification of analytes on a flat sample” describes the use of physically sealed micro-well compartments for analyzing samples contained on a flat substrate. US20100075407 A1 entitled “Ultrasensitive detection of molecules on single molecule arrays” describes digital counting measurements conducted in physically sealed micro-well compartments. WO2012100198 A2 entitled “Methods and systems for performing digital measurements” describes a digital counting measurement conducted by preparing and analyzing arrays of liquid droplets. US20130052649 A1 entitled “Multilayer high density microwells” describes chemically sealed arrays of micro-well compartments for bioanalysis. WO2001061054 A2 entitled “Apparatus and methods for parallel processing of micro-volume liquid reactions” describes the use of chemically sealed capillary arrays for conducting bioassays. WO2014001459 A1 entitled “A method of charging a test carrier and a test carrier” describes the use of capillary arrays for conducting bioassays. WO1998047003 A1 entitled “An analytical assembly for polymerase chain reaction” describes digital counting of oligonucleotides.
Accordingly, there remains a need in the art for improved systems and methods for sealing compartments holding micro-droplets containing material being analyzed.